Ejaculated Semen Samples Research Articles

Successful cryptozoospermia management with multiple semen specimen collection

To determine the prevalence of sperm suitable for intracytoplasmic sperm injection (ICSI) in fresh ejaculated semen samples provided by men scheduled for a microdissection testicular sperm extraction (mTESE) procedure. Secondary objectives included an evaluation of the effect of a short abstinence period on semen quality and ICSI outcomes for men with cryptozoospermia. Retrospective cohort study. Academic medical center. All men were scheduled to undergo a mTESE procedure by a single, high-volume surgeon at an academic center from September 1, 2015, to May 1,2021. Presence of sperm suitable for ICSI in the ejaculate on the day of scheduled mTESE. Prevalence of sperm suitable for ICSI in the ejaculate among previously diagnosed men with azoospermia. Secondary outcomes included changes in semen parameters, clinical pregnancy rate, and live birth rate. Of 727 planned mTESE procedures, 69 (9.5%) were canceled because sperm suitable for ICSI were identified in a fresh ejaculated sample produced on the day of scheduled surgery (typically one day before oocyte retrieval). Overall, 50 men (50/727, 6.9%) used these rare, ejaculated sperm for ICSI. Semen samples obtained with <24 hours of abstinence were more likely to have better motility than the sample initially provided on the day of the planned mTESE. The live birth rate per ICSI attempt using these rare, ejaculated sperm was 36% (19/53). Providing a fresh ejaculated semen sample on the day of mTESE allows nearly 10% of men with azoospermia to avoid surgery with satisfactory ICSI outcomes. Providing multiple ejaculated samples over a short period of time does not adversely affect sperm concentration and may enhance sperm motility in men with cryptozoospermia.

Effect of testicular degeneration on expression of sperm protein at 22kDa in stallions.

Sperm protein at 22kDa has been associated with fertility. The objectives of this study were to determine (1) the localization pattern of SP22 on ejaculated and caudal epididymal equine spermatozoa and in epididymal fluid, and to (2) characterize SP22 protein and mRNA expression in testicular and epididymal tissues in response to heat-induced testicular degeneration. Semen was collected before and after hemi-castration, as well as prior to and following insulation of the remaining testes, and tissue specimens were collected for analysis. Histopathology confirmed degeneration in insulated testes. Ejaculated and epididymal spermatozoa from samples collected prior to insulation of the testicles had a predominant staining pattern of SP22 over the equatorial region. However, the equatorial pattern in the pre-insulation epididymal semen samples was significantly lower than in the pre-insulation ejaculated semen samples (68±3, 81±2.6, respectively). Ejaculated and epididymal samples collected after insulation of the testicles showed a complete loss of staining as the predominant pattern. Western blot analysis verified the presence of SP22 on fresh ejaculated spermatozoa prior to and following heat-induced degeneration, on epididymal spermatozoa after testicular insulation, and in testicular and epididymal tissues. Heat insulation significantly reduced messenger RNA expression in the head of the epididymis and testicular tissues. Immunohistochemistry of the testicular and epididymal tissues pre-heating showed considerably weaker staining than the same tissues post-heating. It was concluded that heat-induced testicular damage causes both loss and relocation of SP22 on the sperm membrane. Future studies are warranted to determine the diagnostic value of these findings.

N-acetyl-l-cysteine and alpha lipoic acid are protective supplement on human sperm parameters in cryopreservation of asthenoteratozoospermia patients.

The aim of this study is to evaluate the beneficial effects of N-acetyl-cysteine (NAC), alpha lipoic acid (ALA) and combination of NAC + ALA supplement in freezing medium on Sperm structural and functional in asthenoteratozoospermia patients. Thirty freshly ejaculated semen samples were cryopreserved with sperm freezing medium (SFM) as control group and three group that SFM supplemented with NAC, ALA and their combination NAC+ ALA. The sperm samples were analysed according to WHO. Mitochondrial membrane potential (MMP), acrosome reaction (AR), antioxidant enzymes and DNA fragmentation were assessed using by Rhodamine123, PSA- FITC ELISA and TUNEL staining respectively. Expression level of NRF2 was assessed by real-time PCR assay. NAC and ALA alone significantly improved sperm motility, viability and DNA fragmentation (p< 0.05). MMP increased in NAC and ALA separately (p< 0.05). While did not affect the amount of sperm morphology and AR (p> 0.05). Antioxidant enzymes significantly difference in NAC and ALA groups (p< 0.05). In addition, NAC and ALA groups showed a significantly higher expression of NRF2 gene compared with other groups (p< 0.05). Our results revealed that the ALA and NAC supplements had a protective effect in cryopreservation process on the structural and functional characteristics of sperm.

O-134 Predictive value of seminal oxidation-reduction potential (ORP) and sperm DNA fragmentation (SDF) analysis for reproductive outcomes of intracytoplasmic sperm injection (ICSI) cycles

Abstract Study question Do seminal oxidation-reduction potential (ORP) and sperm DNA fragmentation (SDF) predict ICSI cycle outcomes? Summary answer Both SDF and seminal ORP have strong prognostic value in predicting good fertilization (≥80%), blastocyst development (≥60%), clinical pregnancy and live birth. What is known already Seminal oxidative stress (OS) has been found to play an important role in various aetiologies of male infertility and is induced by the imbalance between reactive oxygen species (ROS) and antioxidants. OS is a major cause of sperm DNA fragmentation (SDF). It is well established that high levels of seminal OS and SDF are negatively associated with ART outcomes. Previous studies have associated high levels of SDF with an increase risk of miscarriage and a decrease in live birth rate. Study design, size, duration This prospective study included 144 couples who had a fresh autologous ICSI cycle between June 2018 and December 2020. There was no restriction to patients with severe male factor infertility. All cryopreservation and third party assisted reproductive techniques were excluded. Participants/materials, setting, methods This multi-centre study was performed at three ART Clinics in Cape Town, South Africa. All couples included in the study had fresh autologous ICSI cycles with freshly ejaculated semen. Seminal ORP testing was performed using the MiOXSYS system and SDF by means of a fluorescence microscopic TUNEL assay on freshly ejaculated semen samples that were used for ICSI. The ART outcomes evaluated were fertilization rate, blastocyst development rate, clinical pregnancy rate and live birth rate. Main results and the role of chance The study shows that seminal ORP significantly negatively correlates with fertilization rate (r=-0.267; P = 0.0012), blastocyst development rate (r=-0.389; P &amp;lt; 0.0001), pregnancy (r=-0.296; P = 0.0003) and live birth (r=-0.353; P &amp;lt; 0.0001). ROC curve analysis shows significant predictive power for ORP for fertilization (≥80%; AUC=0.652; P = 0.0012), blastocyst development rate (≥60%; AUC=0.762; P &amp;lt; 0.0001), pregnancy (AUC=0.677; P = 0.0002) and live birth (AUC=0.729; P &amp;lt; 0.0001). Comparable results were obtained for SDF. An average cut-off value for ORP of 0.55 mV/106 sperm/mL was calculated. The AUCs between the SDF and ORP results for all reproductive outcome parameters did not differ. For all reproductive outcome parameters, normal sperm morphology shows the lowest predictive power. ORP with male age as confounding factor had significant effects on odds ratios (OR) of fertilization (OR = 0.1446; CI: 0.048-0.430; P = 0.0001) and blastocyst development (OR = 0.2468; CI: 0.095-0.635; P = 0.0029), while it was not significant for pregnancy and life birth. Multivariate logistic regression with fertilization, blastocyst development, pregnancy and live birth as dependent parameters and high/low seminal ORP, primary and secondary infertility, unexplained infertility, polycystic ovaries, endometriosis, tubal factor myomectomy, ovarian insufficiency and advanced maternal age as independent variables showed overall model fits (P &amp;lt; 0.0002) with a significant (P &amp;lt; 0.0084) influence of ORP on all reproductive outcome parameters. Limitations, reasons for caution Since only ICSI patients were included in this study, this might have contributed to the fact the cut-off value of ORP is lower than the published cut-off. A further limitation is the use of a fluorescence microscopic TUNEL assay which drastically limited the number of sperm evaluated. Wider implications of the findings This is the first study showing predictive value of seminal ORP for reproductive outcome including pregnancy and live birth. The method is easy and much quicker than current methods for the determination of SDF. Hence, it can be used a point of care method. Trial registration number Not applicable

P-063 Multivariate analysis identifies oxidative stress levels and acrosome status of motile spermatozoa as independent predictors of motile sperm hyaluronan binding ability

Abstract Study question Is it possible to predict motile sperm hyaluronan binding ability (HBA) from motile sperm functional parameters? Summary answer Using multivariate analysis, oxidative stress levels and acrosome status of motile spermatozoa, derived from semen swim-up, were identified as predictors of sperm HBA. What is known already Semen analysis is a gold standard practice for fertility evaluation. However, motility and morphology of spermatozoa are not always indicative of the molecular makeup of sperm cells. In this context, several sperm functional tests were developed to enhance our understanding of spermatozoa biology. Interestingly, several studies have shown that the plasma membrane of mature sperm cells contain hyaluronan binding sites. HBA was associated with intact acrosome, decreased DNA damage, and high viability. Nevertheless, it has not been demonstrated yet if hyaluronan binding ability can be predicted solely by swim-up derived- motile spermatozoa functional parameters. Study design, size, duration A prospective study was performed at Al-Hadi Medical Center, beirut, Lebanon, between January and July 2020. Freshly ejaculated semen samples obtained from infertile men with concentration of ≥ 20 x 106/ml and with total sperm motility of ≥ 30% were included. The primary outcome was to assess if the motile sperm functional parameters may influence HBA. A sample size of 54 was calculated using power = 85% and alpha = 5% for multiple linear regression. Participants/materials, setting, methods 54 semen samples were assessed according to WHO guidelines (2010). Thereafter, samples were processed using the semen swim-up technique. Supernatants containing motile spermatozoa were collected for further analysis. In motile spermatozoa-enriched fractions, chromatin integrity was assessed using toluidine blue (TB) staining. Oxidative stress levels were detected using a nitroblue tetrazolium (NBT) test. Acrosome status was determined using triple staining technique. HBA was evaluated via a commercially available hyaluronan binding assay (HBA® Assay). Main results and the role of chance In the motile spermatozoa-enriched fractions, mean ± standard deviations for concentration, progressive motility, non-progressive motility and normal morphology were 26.17±11.48, 76±18.97, 20.94±12.61 and 0.75 ± 1.4, respectively. In addition, mean ± standard deviations for TB + cells, NBT + cells, living spermatozoa with intact acrosome, and HBA were 16.42 ± 13.4, 57.4 ± 9.4, 93.9 ±3.2, and 48.98±11.95, respectively. The values of the HBA had a non-normal distribution; therefore, these values were first linearized by taking the logarithm of the outcome. Afterwards, multiple linear regression (MLR) was used to predict the outcome. The overall regression of the final model was statistically significant (R2 = 0.346), F(4.43) =5.962, p = 0.001. The results of the final MLR model indicated that a unit increase in non-progressive motility (%) (p = 0.002) and NBT + cells (%) (p = 0.009) increased the HBA by 1.505 units and 1.409 units, respectively. However, a unit increase in dead spermatozoa with unreacted acrosome (%) (p = 0.032) and living spermatozoa with reacted acrosome (%) (p = 0.026) decreased the HBA by 1.317 units and 1.342 units, respectively. Limitations, reasons for caution This is an interim study and these results should be taken as preliminary. In order to confirm these findings, further studies including larger sample size and motile spermatozoa derived from semen samples with abnormal parameters (such as severe asthenozoospermia, leukocytospermia, severe oligozoospermia) are required. Wider implications of the findings Several studies indicated that using damaged spermatozoa may affect the intracytoplasmic sperm injection (ICSI) outcomes. In addition, using damaged spermatozoa during fertilization may predispose the offspring to various diseases later in life. These results may be considered as an impetus to reconsider sperm selection during ICSI. Trial registration number Not applicable

The benzene impact on programmed death of sperm cell

Introduction. Benzene and its derivatives enter organism from the natural environment are the sources of constant health danger, including reproductive system disorders. Purpose of the study. In vitro study of the modifying effect of benzene on the indices controlling the apoptosis of sperm cells. Materials and methods. The study includes data on 27 men from 41 to 51 years of age, apparently healthy, not in contact with harmful factors, and living in relatively favourable conditions. Using flow cytometry in the ejaculated semen, we determined CD25+ and CD95+ markers, p53, bax, caspase-3 activity, percentage of AnnexinV-FITC+PI-, and AnnexinV-FITC+PI+-spermatozoa. The seminal fluid samples were incubated with benzene at a concentration of 0.001 µg/ml (experimental samples) for 72 hours at 370C to evaluate the effect of aromatic hydrocarbons on the indices controlling the apoptosis. Sperm samples incubated under identical conditions without benzene addition were used as control samples. Results. p53 content, caspase-3 activity, and the number of dead male germ cells (AnnexinV-FITC+PI+-spermatozoa) were found to decrease statistically significantly (p=0.023-0.026) in experimental semen samples after the addition of benzene (by 50% of the initial levelon average). At the same time, the addition of benzene to the ejaculated semen samples did not change the early activation profile of gametes (according to CD25+ criteria), cells readiness to start FAS-dependent apoptosis (CD95+), and the number of spermatozoa marked for death by apoptosis (AnnexinV-FITC+PI- - spermatozoa). Conclusion. The results of immunological testing demonstrated that in vitro system benzene inhibits apoptosis and interferes with gamete life cycle . On the example of benzene, it was been demonstrated that haptenic contamination could alter sperm fertility associated with an imbalance of proapoptotic factors and impair ed male reproductive system function. The indices of programmed cell death bax, p53, caspase-3, CD25-positive, FAS-positive, AnnexinV-FITC+PI--sperm, and AnnexinV-FITC+PI+-sperm in the ejaculate are recommended for diagnosis and monitoring of sperm fertility disorders.

Collecting semen samples at home for IVF/ICSI does not negatively affect the outcome of the fresh cycle

Research questionDoes the site of semen collection influence IVF/intracytoplasmic sperm injection (ICSI) cycle outcome? DesignRetrospective study performed at the University Medical Centre Ljubljana, including all stimulated and modified natural IVF/ICSI cycles (with at least one oocyte retrieved) performed in 2019 with fresh ejaculated semen samples. IVF/ICSI cycle outcomes, in terms of oocytes, embryos and pregnancy rates according to site of semen sample collection (at home or at clinic) were evaluated. ResultsSamples collected at clinic had significantly lower sperm concentration (median [interquartile range, IQR], 50 [20–100] million/ml versus 70 [30–100] million/ml, adjusted odds ratio [OR] 0.001, 95% confidence interval [CI] 1.574 × 10–6 to 0.196, P = 0.012) and motility (60 [50–70]% versus 70 [50–70]%, adjusted OR 0.034, 95% CI 0.002 to 0.563, P = 0.018, adjusted for age). There was no difference in total sperm count, semen volume or sperm morphology, or women's age (36 [32–39] versus 36 [33–39] years) and men's age (37 [34–41] versus 38 [34–42] years), between semen samples collected at clinic versus at home. When all IVF/ICSI cycles were analysed together using generalized estimating equation analysis, no significant difference in cycle outcomes attributed to site of semen sample collection was observed. There were also no significant differences in cycle outcomes when only first cycles were analysed. ConclusionsCollecting semen samples at home has a positive effect on sperm quality (sperm concentration and motility were higher), but no significant differences in cycle outcomes are observed when these samples are used in IVF/ICSI cycles. Therefore, it is suggested that collecting semen samples at home for IVF/ICSI procedures is safe and has no negative effect on treatment outcomes.

Genomic Sequencing Reveals the Diversity of Seminal Bacteria and Relationships to Reproductive Potential in Boar Sperm.

A number of emerging studies suggest that pathogenic microorganisms in semen may cause a decline in the reproductive potential of spermatozoa, and the bacterial diversity and profile of ejaculated boar semen in different seasons are currently unknown. To explore the bacterial composition and changes in ejaculated boar semen from winter and summer, and the underlying mechanism of decline in sperm quality and fertility capacity in summer, 120 ejaculated semen samples were examined for bacterial communities using genomic sequencing technology, and the associations between microbial composition and sperm reproductive potential were investigated. The results showed that Proteobacteria (57.53%), Firmicutes (31.17%), Bacteroidetes (4.24%), and Actinobacteria (3.41%) are the dominant phyla in the ejaculated semen, and the dominant genera were Pseudomonas (34.41%) and Lactobacillus (19.93%), which belong to the phyla of Proteobacteria and Firmicutes, respectively. Interestingly, the higher diversity of bacteria in ejaculated semen of winter differs from that of summer semen, potentially due to seasonal changes related to changes in semen quality and sperm fertilizing capacity. Furthermore, the highly abundant Lactobacillus in winter samples were positively associated with sperm quality and reproductive performance obtained from sows inseminated with such semen samples, while in contrast, the highly abundant Pseudomonas in summer samples was negatively associated with sperm quality and reproductive potential. Additionally, our results strongly indicated that Lactobacillus is not only a potential probiotic for semen quality and fertility potential but also beneficial for restraining the negative influence of Pseudomonas. Overall, our findings significantly contribute to the current understanding of the phenotypes and etiology of male “summer infertility,” and may represent a frontier in male reproductive disorders and possible early prevention against pathogenic bacteria.

Supplementation of freezing media with alpha lipoic acid preserves the structural and functional characteristics of sperm against cryodamage in infertile men with asthenoteratozoospermia

The aim of this study was to examine the effects of alpha lipoic acid (ALA) supplementation during semen cryopreservation on the sperm quality, chromatin integrity, oxidative stress, and expression level of BAX, BCL2, HSP70 and iNOS genes in semen samples obtained from infertile men with asthenoteratozoospermia. MethodsTwenty freshly ejaculated semen samples were cryopreserved with sperm freezing medium supplemented with 0.00, 0.02, 0.05, 0.1, 0.5, and 1 mmol/mL of ALA. The samples were analyzed according to the WHO guidelines before and after freezing. Sperm ROS production level, DNA fragmentation and cryo-capacitation were assessed using flow cytometry, TUNEL assay and chlortetracycline (CTC) test, respectively. Expression level of stress protein (HSP70), pro-apoptotic Bax, anti-apoptotic Bcl-2, and iNOS genes was assessed by real-time PCR assay. ResultsThe effective concentrations of ALA (0.02 and 0.5 mM) significantly improved the motility, viability and morphology of the frozen-thawed sperms compared to the control group treated with 0.00 mM of ALA. During cryopreservation, treatment of semen with 0.02 mM of ALA, as the optimal concentration, significantly decreased DNA fragmentation and oxidative stress level (P < 0.05), protected the acrosome integrity, and led to insignificant reduction in BAX gene expression level and significant increase in expression level of BCL2, HSP70, and iNOS genes compared with control group. ConclusionOur findings revealed that the adding ALA to semen samples obtained from infertile men with asthenoteratozoospermia plays a significant protective role against cryodamage by preserving the sperm functional parameters.

No evidence of severe acute respiratory syndrome–coronavirus 2 in semen of males recovering from coronavirus disease 2019

ObjectiveTo describe detection of severe acute respiratory syndrome (SARS)–coronavirus 2 (CoV-2) in seminal fluid of patients recovering from coronavirus disease 2019 (COVID-19) and to describe the expression profile of angiotensin-converting enzyme 2 (ACE2) and Transmembrane Serine Protease 2 (TMPRSS2) within the testicle.DesignObservational, cross-sectional study.SettingTertiary referral center.Patient(s)Thirty-four adult Chinese males diagnosed with COVID-19 through confirmatory quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) from pharyngeal swab samples.Intervention(s)None.Main Outcome Measure(s)Identification of SARS-CoV-2 on qRT-PCR of single ejaculated semen samples. Semen quality was not assessed. Expression patterns of ACE2 and TMPRSS2 in the human testis are explored through previously published single-cell transcriptome datasets.Result(s)Six patients (19%) demonstrated scrotal discomfort suggestive of viral orchitis around the time of COVID-19 confirmation. Severe acute respiratory syndrome–CoV-2 was not detected in semen after a median of 31 days (interquartile range, 29-36 days) from COVID-19 diagnosis. Single-cell transcriptome analysis demonstrates sparse expression of ACE2 and TMPRSS2, with almost no overlapping gene expression.Conclusion(s)Severe acute respiratory syndrome–CoV-2 was not detected in the semen of patients recovering from COVID-19 1 month after COVID-19 diagnosis. Angiotensin-converting enzyme 2–mediated viral entry of SARS–CoV-2 into target host cells is unlikely to occur within the human testicle based on ACE2 and TMPRSS2 expression. The long-term effects of SARS–CoV-2 on male reproductive function remain unknown.

Ejaculated compared with epididymal stallion sperm vitrification

The aim of this study was to evaluate the effect of trehalose and lactose extenders on ejaculated and epididymal stallion sperm vitrification. Ejaculated semen samples were collected from seven fertile stallions, and cauda epididymis samples were collected from ten stallion carcasses after slaughter. Both the ejaculated and the epididymis samples were diluted and vitrified using INRA 96® and bovine serum albumin as well as trehalose or lactose. As a control, ejaculated and epididymal samples were collected and frozen using the conventional method. Vitrification was performed by immersing sperm suspensions directly in LN2. After thawing or devitrification, there was assessment of samples for sperm motility using computer-assisted analysis. Viability was assessed using SYBR-14 and propidium iodide (PI) and acrosome integrity by fluorescein using isothiocyanate combined with peanut agglutinin (FITC-PNA) and PI. Epididymal sperm vitrification with trehalose (EPT) or lactose (EPL) resulted in greater progressive sperm motility than sperm of the control sample (EPC). After post-thaw/devitrification of sperm in the EPT group, sperm motility was greater (P<0.001) compared to that using EPL (50.72 ± 5.09% compared with 34.21 ± 3.02%). The results from assessment of ejaculated sperm samples after undergoing the vitrification process indicated cells were less viable (P<0.001) than the control (EJC) sample. In conclusion, vitrification of epididymal stallion sperm using trehalose might be a beneficial alternative for the long-term storage of sperm samples with great economic value. Spermatozoa from vitrified ejaculates of stallions, however, had lesser motility and viability rates than samples subjected to conventional freezing.

Effect of butylated hydroxytoluene on quality of pre—frozen and frozen buffalo semen

Objective: To clarify the antioxidant effect of butylated hydroxytoulene (BHT) at different concentrations on cooled and post frozen semen diluted in tris-citrate-fructose egg yolk glycerol and lecithin -based extenders. Methods: Forty ejaculates were harvested from four buffalo bulls by means of the artificial vagina. Ejaculated semen samples were diluted with each of the tris citrate-fructose egg yolk glycerol and lecithin-based extender diluents. The semen samples diluted with each of the two extenders were added to pre-warmed dried test tubes containing BHT (prepared in ethanol) to get concentrations at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mM/mL BHT. These ingredients were put at 37 °C for 5 min to allow the proper BHT spermatozoal permeation. The diluted semen samples were cooled to 5 °C and then frozen to -196 °C in 0.25 mL ministraws before dipping in liquid nitrogen pending its evaluation. Sperm motility, viability, morphology, intact acrosome and membrane integrity were tested. Visual motility was tested using a high power ordinary microscope (at 400 ×) with closed circuit television, and sperm concentration was tested using Neubauer haemocytometer and abnormality % using eosin-nigrosin stain. Spermatozoal membrane integrity was tested using the hypo-osmotic swelling test. The sperm with swollen twisting tail was normally intact. Sperm acrosomal integrity % was tested as mentioned by Watson. Results: Addition of BHT improved (P<0.01) progressive motility, viability, morphology and acrosome as well as plasma membrane integrities at 0.5-2.0 mM/mL depending upon types of used extenders and stages of pre-and post-freezing process. Higher levels of 2.5 and 3.0 mM/mL BHT had a deteriorating (P<0.01) result if compared to the control and all extenders assayed. Conclusions: BHT addition at lower concentration can improve pre-frozen and post-thawed buffalo sperm quality.

Investigating the role of sperm-specific RNA to screen men with unexplained infertility

To identify the compartmentalization of candidate gene clusters within the human spermatozoon that contribute to spermatozoal fertilization capacity and embryo developmental competence. Sequencing and differential expression of sperm-specific RNAs in men with unexplained infertility. Consenting men screened for infertility donated their ejaculated semen samples during a 15-month period. Donors with proven fertility served as controls. RNA was isolated from 25x106 human spermatozoa using a commercially available spin column kit. Spermatozoal RNA concentration was subsequently assessed. Illumina stranded RNA sequencing (RNA-Seq) library preparation was used to construct paired-end libraries. Pilot sequencing was expanded to 50-60M reads at 2x76bp. RNA expression was calculated in fragments per kilobase of transcript per million mapped reads (FPKM). Differentially expressed RNA transcripts were identified and classified into gene clusters based on sperm-specific components i.e., the acrosome, nucleus, mid-piece, and flagellum. The expression of these transcripts was compared with standard semen parameters, and between the study and control groups. Thirty-one men with a mean age of 39.6±5 years had the following semen parameters: 46.3±19 x 106/mL concentration and 44.8±14% motility. The age of the female partner in the study and control groups was comparable. Differentially expressed RNA transcripts between the study and control groups were grouped into sperm-specific clusters: DPY19L2 and ATP6V1E2 (acrosomal); APLF, CYUB5R4, ERCC4, MORC1, PIWIL1, TNFRSF21, TSSK6 and H1FNT (nucleus); ADCY10, SMCP, PLK4 and AGPAT2 (mid-piece); and, AKAP4 and CATSPER1 (flagellum). There was a strong correlation between the expression of ATP6V1E2 and fertilization rates (R2 0.63; P<0.001) in men who underwent ICSI. A positive correlation between AKAP4 and motility was also noted (R2 0.40; P=0.01). The gene cluster localized to the nucleus was significantly under-expressed in the study group when compared to the control group (P<0.001). Interestingly, the expression of APLF, CYUB5R4, ERCC4, and TNFRSF21 was found to be higher in men <40 years (47.2±15 FPKM) compared to those >40 years (13±9 FPKM; P=0.02). Functional assessment of the gene cluster specific to certain compartments of the human spermatozoon may supplement standard semen analysis and serve as a vital tool in the diagnosis of unexplained infertility. This may further facilitate tailoring of assisted fertility treatments, thereby reducing the time to pregnancy.

Determining embryo developmental competence by measuring expressivity of the paternal genome

To profile the transcriptome of men with unexplained infertility and to identify key sperm-specific RNAs that contribute to post-fertilization embryonic developmental competence. Sequencing and differential expression of sperm-specific RNAs in men with unexplained infertility. Next generation sequencing (RNA-Seq) was performed on ejaculated semen samples provided by consenting men undergoing infertility screening. All men had normal semen parameters and their female partners <35 years had normal infertility screening. RNA was isolated from 25x106 spermatozoa using a hybrid isolation protocol with TRIzol® Reagent (ThermoFisher Scientific, USA) and a spin column kit (RNeasy Mini Kit, QIAGEN, Germany). Pilot sequencing was carried out at 2x36bp to determine library quality and expanded to 50-60M reads at 2x76bp utilizing an Illumina platform (NextSeq 500). Expression values were calculated in FPKM (Fragments Per Kilobase of Exon Per Million Fragments Mapped). The expression of RNA transcripts was compared between men who were and were not able to achieve conception with intracytoplasmic sperm injection (ICSI). The expression profiles of these RNA transcripts were then compared to embryo development parameters. The 25 consenting men had the following semen parameters: 37.3±17 x 106/mL (concentration), 46.6±10% (motility), and normal morphology. The average concentration of RNA extracted was 14.3±6 ng/μL. The age demographics were comparable among couples who conceived i.e., fertile (men=37.6±3 years; women=34.8±3 years) and did not conceive i.e., infertile (men=38.3±5 years; women=33.6±7 years) with ICSI. The fertilization rates with ICSI and number of embryos transferred were also similar. RNA expression analysis revealed 25 differentially expressed genes between the groups (P<0.001): 10 overexpressed and 15 underexpressed. The large majority of differentially expressed sperm-specific RNAs (23/25) were particularly important for the development of embryos from the 1-cell to 4-cell stage. A higher embryonic fragmentation rate (14.3%) was noted in the infertile group, which corresponded to a higher expression of BOK RNA transcripts in the sperm of infertile patients (P<0.001). PLK4 and BUB1, which dictate chromosome stability and mitotic segregation by regulating centrosome development, had decreased expression in the infertile group (P<0.001). Transcriptome profiling of human spermatozoa using RNA-Seq can reveal genes products that are critical to embryonic development. Our results suggest that sperm-specific RNAs are vital for embryonic development between the 1-cell to 4-cell stage.

Melatonin receptors MT1 and MT2 are expressed in spermatozoa from several seasonal and nonseasonal breeder species

Melatonin is a ubiquitous and multipurpose molecule, and one of its roles is to regulate reproduction in some seasonal mammals. Our group has previously reported the variation in the melatonin levels in ram seminal plasma along the year and identified MT1 and MT2 receptors in ram spermatozoa. The objective of this study was to elucidate whether the presence of melatonin receptors (MT1 and MT2) in the sperm plasma membrane, and melatonin in the seminal plasma is related to seasonal breeding. For this purpose, the presence of melatonin receptors and the levels of melatonin in seminal plasma have been examined in several species: donkey and stallion as long-day breeders; red deer as a wild, short-day, highly seasonal breeder (epididymal spermatozoa); bull as a conventional nonseasonal breeder; boar as a seasonal breeder under management techniques; and dog as possible a seasonal breeder not regulated by melatonin. We have detected measurable levels of melatonin in the seminal plasma of all ejaculated semen samples (from donkey, stallion, boar, bull, and dog). Also, and for the first time, we have demonstrated the presence of MT1 and MT2 melatonin receptors in the spermatozoa of all these species, regardless their type of reproduction or sperm source (ejaculated or epididymal), using indirect immunofluorescence techniques and Western blotting. Our findings suggest that melatonin and melatonin receptors may be universally distributed in the reproductive system of mammals and that the sperm melatonin receptors cells may not be necessarily related with seasonal reproduction. Furthermore, the presence of MT1 at the cytoplasmic droplet in immature ejaculated stallion spermatozoa found in one sample and epididymal red deer spermatozoa suggests that melatonin may be involved in specific functions during spermatogenesis and sperm maturation, like protecting spermatozoa from oxidative damage, this activity being mediated through these receptors.

Rapid thawing and stabilizing procedure improve postthaw survival and in vitro penetrability of boar spermatozoa cryopreserved with a glycerol-free trehalose-based extender

Thawing process is important in semen cryopreservation as it brings back the sperm cell to physiologic temperature reactivating the metabolism. Aims of the present study were to evaluate survival rate and in vitro penetrability of boar frozen spermatozoa after rapid and transient thawing at a high temperature followed by a warming procedure at 39 °C. Ejaculated semen samples were diluted in an egg yolk–based glycerol-free extender containing 100-mM trehalose and then cryopreserved in 0.5-mL straws according to a common protocol. In experiment 1, when temperature inside the straws was monitored after thawing at 40 °C, 60 °C, 70 °C, and 80 °C, the calculated average warming rate in the straws from −196 °C to 15 °C was much faster when thawed at 70 °C and 80 °C than at 40 °C (P < 0.01). The warming temperature rate inside the straw was 7 to 12 folds faster during the first 2 seconds than the second 2 seconds after immersing in high temperatures. In experiment 2, when frozen straws were thawed at 80 °C for 9 seconds, the viability, motility, and acrosomal integrity were significantly improved (P < 0.05), as compared with controls (at 39 °C). In experiment 3, frozen straws were thawed at 39 °C, 60 °C, 70 °C, and 80 °C for 60, 10, 8, and 6 seconds, respectively, and then maintained at 39 °C for 0, 50, 52, and 54 seconds. Higher viability, motility, mitochondria membrane potential, and acrosome integrity were observed (P < 0.05) when frozen straws were thawed at 70 °C for 8 seconds and then maintained at 39 °C for 52 seconds as compared with the control (39 °C for 60 seconds). In experiment 4, in vitro penetrability of frozen spermatozoa thawed at 70 °C for 8 seconds and maintained at 39 °C for 52 seconds was higher than that of controls. In conclusion, the rapid transient thawing at 70 °C for 8 seconds followed by stabilizing procedure at 39 °C for 52 seconds maintained the viability, motility, mitochondria membrane potential, acrosome integrity, and in vitro penetrability of spermatozoa frozen in a glycerol-free trehalose extender and recommended as an optimum thawing conditions.

New method to estimate the possibility of natural pregnancy using computer-assisted sperm analysis

The World Health Organization (WHO) criteria, which include percent motility and sperm concentration, are the only criteria for evaluating sperm quality and conception ability. However, these criteria are insufficient to evaluate the possibility of natural pregnancy. Thus, an index that can directly evaluate the possibility of a natural pregnancy is necessary. A new sperm energy theory without approximation was developed to assess the possibility of natural pregnancy based on mechanical sperm energy. Sperm motility parameters were measured using computer-assisted sperm analysis (CASA) in 129 ejaculated semen samples from 50 men in couples diagnosed with infertility, in which no abnormalities were found in women (sterile group), and 157 ejaculated semen samples from 57 men who had already fathered children in natural pregnancies (control group). A total of 129 subjects were selected from the control group and classified as the fertile group in order of the sample measurement date. The sperm energy index (SEI) and mean sperm energy index (MEI) were accurately obtained according to the methods described by the new sperm energy theory. SEI reflects total mechanical energy of the sperm in a visual field during CASA measurements. MEI reflects the mean mechanical energy of one sperm in a measurement field. All subjects with (MEI)/(SEI) > 2 were assigned to the sterile group. The larger the SEI, the higher was the probability of predicting fertile subjects. The probability of predicting fertile subjects was approximately 60% with a SEI of > 0.5, 70% with a SEI of > 1, 80% with a SEI of > 3, and 90% with a SEI of > 6 in cases where (MEI)/(SEI) is < 2. The data support the view that this novel method can be used to estimate the possibility of a natural pregnancy.

Bacterial contamination of ram semen, antibiotic sensitivities, and effects on sperm quality during storage at 15 °C

This study was designed to determine the degree and type of bacterial contamination of ejaculated semen samples in fertile rams and its consequences on sperm quality during storage. In experiment 1, 68 ejaculates from 36 rams were divided into two aliquots, one of which was used for bacterial culture, while the other one was diluted, stored at 15 °C and assessed for plasma membrane integrity and motility at 0, 24 and 48 h after dilution. From the 68 ejaculates, 66 were positive for aerobic bacteria, including 20 species of bacteria from 14 genera. The most frequently isolated bacteria were Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Staphylococcus epidermis, and Staphylococcus aureus species. These 5 bacteria were present in 97% of all contaminated samples. All contaminant bacteria were found to be sensitive to gentamicin and to ceftiofur, with variable percentages of resistance to the other antibiotics evaluated. In samples with total enterobacteria count lower than 100 colony-forming units (CFU)/ml, higher proportions of motile and progressive sperm and higher velocities of spermatozoa were observed at different times during storage. In experiment 2, pure cultures of the most frequently isolated bacteria were individually added to fresh semen samples of low contamination and tested for their effects on sperm quality during storage at 15 °C. Semen with E. coli showed a drastic reduction in motility, velocity and viability during storage. This reduction was also significant, but less drastic, in semen with E. cloacae and P. mirabilis, whereas it was partial and less pronounced in the other groups ( S. epidermidis and S. aureus). In conclusion, the contamination of ram semen with enterobacterial species reduced sperm quality during storage at 15 °C, and the antibiotics gentamicin and ceftiofur showed the higher antimicrobial activities.

Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa

Appropriate semen processing and assessment are critical for successful infertility treatment. We investigated whether laboratory procedures including semen preparation and incubation affect sperm DNA integrity. A total of 153 infertile men were involved. Conventional semen parameters and sperm chromatin structure assay (SCSA) parameters, that is, DNA fragmentation index (%DFI) and high DNA stainability (%HDS), were assessed on the fresh ejaculated semen samples, which were treated and incubated under different conditions. Negative correlations were identified between the %DFI and sperm concentration, motility, progressive motility and morphology. A lower percentage of DFI was detected in spermatozoa when density gradient centrifugation (DGC) was followed by swim-up treatment in comparison with DGC alone (P < 0.01). Although the %DFI increased in a time-dependent manner with incubation both at room temperature (RT) and at 37 degrees C in air, the %DFI after 24 h at RT was significantly lower than that at 37 degrees C (P < 0.05). Incubation with 5% CO2 was effective in maintaining sperm motility (P < 0.01); however, it induced further elevation of %DFI (P < 0.001). Thus, sperm DNA damage was associated with longer incubation periods. Interestingly, common culture conditions, such as maintaining pH and temperature, compromised the sperm DNA integrity.

Assessment of fertility by sperm mechanical energy using computer-assisted sperm analysis system.

It is known that the energy distribution of sperm obeys the following equations and the total mechanical energy in a sperm population is expressed as nKλ × a constant. nKλ/102 is defined as the sperm energy index (SEI). [Formula: see text] Here, P is the existing probability density of sperm, t is the square of amplitude of lateral head displacement, λ is the mean of t, n is the number of motile sperm in semen in a measurement field, and K is a constant determined for each semen sample. These values can be obtained with the use of computer-assisted sperm analysis (CASA). Sperm motility parameters were measured using CASA in 163 ejaculated semen samples from 47 infertile men (infertility group), and 162 ejaculated semen samples from 45 fertile men (natural pregnancy group). Kλ is defined for the present investigation as the mean energy index (MEI). SEI and MEI were obtained according to the methods described in sperm energy theory. There were no natural pregnancy subjects with SEI < 0.5. All subjects with MEI > 2.0 and SEI > 1.0 were in the natural pregnancy group. An assessment of fertility was possible by using the sperm energy index.